Diphtheria toxin is a 58,000 dalton protein that has the ability to translocate across lipid bilayers. The toxin is internalized into acidic endosomes where the toxin B subunit undergoes a conformational change resulting in its insertion into the endosomal membrane and the transfer of the A subunit across the membrane into the cytosol. Our studies are directed at better understanding the mechanism by which the toxin B subunit can undergo conformational changes resulting in translocation across the endosomal membrane. The toxin B subunit contains four hydrophobic domains proposed to be involved in translocation. Proline residues are found in high representation and in conserved locations in these hydrophobic domains similar to the arrangement found in transport proteins that span the membrane multiple times. One of these conserved prolines (position 345) was changed to Glu or Gly using a recombinant PCR technique with mismatched primers. These mutants were transcribed and translated in vitro. The mutant toxins demonstrated comparable binding activity and enzymatic activity to the native protein but were 100-fold less toxic to cells in tissue culture reflecting a 100-fold reduction in translocation activity. These results demonstrate the critical role of Pro 345 in membrane translocation.